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A new Scanbox panel allows users to define arbitrary changes in (x,y,z) position over time (frames) which are then executed by Knobby (version 2 only) while imaging.

Each entry define changes in x, y and z (in micrometers) relative to the present position and the frame number at which they will take place.

The “mem” column allows one to specify one of the stored absolute coordinates instead (memory locations are coded A=1, B=2, C=3). If a memory location is defined the other entries are ignored and the position in the referenced memory is used instead.

This mechanism extends the z-stack functionality to include the ability to tile a sample and brings back the control window to one of the panels in Scanbox (as opposed to being controlled in Knobby’s screen). The Knobby table is also saved in the info.knobby_table variable.

Paths can be computed offline and stored in a Matlab file that can be loaded. The example below shows knobby moving the sample along a circular path.

Update [ 7/3/17]: You will now note an additional checkbox in the Knobby Scheduler panel called “Return”. When you arm knobby scheduler and check this box the microscope will automatically return to the initial position after imaging is done. This feature uses the Store/Recall C function for functionality. Anything stored previously in C is going to be erased if you use this feature.

In the ports panel of the Bootloader host you will see a USB Human Interface Device listed immediately. Select the device and click the Download button at the top left of the Bootloader Host Window (it is the one with an arrow pointing down). Note: you ave 20 seconds to upload the new firmware (from the time you issued the Matlab commands), otherwise Scanbox will continue booting normally. The Log panel in the Bootloader host will show if the programming of the box was successful. You can now quit the Bootloader host.

The new firmware version should now show up on the Scanbox LCD display.

Now we need to update Knobby. To do so, simply go to the Matlab window and type “knobby_update”. Wait for Knobby to update.

The last release of Scanbox provides a mechanism to update the firmware in Knobby and provides a few fixes to the existing version. We will be using this mechanism to deploy future improvements.

The present version fixes a bug that incorrectly moved the objective to its original position when the X, Y, or Z axes where moved right after a Zero with vertical alignment was performed. It also provides a way to cancel a Zero/Store/Recall function if they were triggered by mistake. To cancel you simply have to touch the same button once again.

The new version also adjusts the hit areas on the touchscreen for the Lock/Unlock button and the XYZ menu selection for the Zero command, which were slightly off causing some unnecessary frustration.

To update Knobby’s firmware proceed as follows.

After downloading the latest Scanbox versions use pathtool in Matlab to ensure all the sub-directories within the distribution are added to the path. Then within Matlab, type “knobby_update”. If everything look Ok you will see something like the output below. Your Knobby will reboot and you can then start Scanbox and start using it.

To take advantage of the new features in the latest release of the Yeti firmware you should start by getting rid of the half-wave plate control of the laser power. To do so start by removing the motor and half-wave plate from the optical path feeding the laser beam directly into the Pockels cell as shown in the picture below. Make use of a detector card and/or power meter to check for alignment.

Make sure the Pockels cell is aligned so that there is minimum transmission when the reading in the front panel is zero. The power of the laser in this new arrangement is controlled by a slider in the Chameleon panel, which provides control from 0% to 100% of the power.

The software takes into account the fact that at 920nm the relationship between power and control voltage is non-linear, so you should get an approximate linear control of the power using the slider. This new feature provides for an absolute control of the power across experiments (as compared to the the relative position of the wave-plate in the original design). You should also get increased power, as the new setup avoid loses via multiple reflections.

Using the new firmware you also have control over the size of the dead-band at the edges of the scanning. A new variable in the configuration file, sbconfig.deadband, can be changed by the user. The two numbers of the vector reflect the size of the dead-band at the left and right margins of the scan in percent of the total number of samples before spatial correction.

The reason for this changes is that this new setup also allows the software to rapidly change the power, a feature that will be needed when the Optotune lens is incorporated for fast z-scanning in the near future. If you plan to use the optotune, you should be upgrading to this new firmware as soon as possible.

Updating the firmware

The lastest firmware is located in the Scanbox/drivers/ subdirectory.

To update the driver start the Bootloader Host application (look under the Cypress Programs Folder).

Select the new Firmware file you want to upload (the one in the drivers/ directory), power cycle Scanbox to get it into boot loader mode. Once you turn the box on you will have 20 sec to upload the new firmware (once the time elapses Scanbox will start running the existing firmware).

Select the USB Human Interface Device that will appear in the ports panel, and click the download button (the one highlighted in blue).

If everything works you will get a message that the firmware was successfully uploaded (it should take 3-4 seconds to complete). Once the new software is uploaded Scanbox should be ready to use.

If you have trouble with the firmware updating process just let me know…

Note: You can download the PSoC suite from here but make sure you select “Custom” installation (instead of “Typical”) and select the “bootloader host” only for installation (otherwise you will be installing the whole suite and it will take a very large amount of time/space).